This invention relates to the detection of biomolecules. Specifically, the invention relates to an apparatus and methods for efficient, high-throughput electrical or electrochemical detection of biomolecules. In addition, the invention relates to such apparatuses having manipulation electrodes associated with test sites for enhancing concentration or reaction of a biomolecule at a test site, with or without providing detection electrodes at the test sites. In embodiments having detection electrodes, the manipulation electrodes increase the occurrence of a desired bio-conjugation event at a test site, while the detection electrodes detect the occurrence of the desired bio-conjugation event. Methods for using the apparatuses of the invention, particularly for analysis of nucleic acids, are also provided.
2. Background of the Invention
A number of commonly-utilized biological applications, including for example, diagnoses of genetic disease, analyses of sequence polymorphisms, and studies of receptorligand interactions, rely on the ability of analytical technologies to readily detect events related to the interaction between biomolecules. These detection technologies have traditionally utilized fluorescent compounds or radioactive isotopes to monitor such interactions. For example, Potyrailo et al., 1998, Anal. Chem. 70: 3419-25, describe an apparatus and method for detecting interactions between immobilized fluorescently-labeled aptamers and peptides. There are, however, significant disadvantages associated with the use of radioactive or fluorescent labels to track interactions between biomolecules, including heightened health risks and increased experimental cost and complexity.
Methods for electrical or electrochemical detection of molecular interactions between biomolecules have provided an attractive alternative to detection techniques relying on radioactive or fluorescent labels. Electrical and electrochemical detection techniques are based on the detection of alterations in the electrical properties of an electrode arising from interactions between one group of molecules attached to the surface of an electrode (often referred to as xe2x80x9cprobexe2x80x9d molecules) and another set of molecules present in a reaction mixture (often referred to as xe2x80x9ctargetxe2x80x9d molecules) contacted with the electrode. Methods and devices related to electrical or electrochemical detection of biomolecules are disclosed in U.S. Pat. Nos. 4,072,576, 4,098,645, 4,414,323, 4,840,893, 5,164,319, 5,187,096, 5,194,133, 5,312,527, 5,532,128, 5,591,578, 5,653,939, 5,670,322, 5,705,348, 5,770,369, 5,780,234, 5,824,473, 5,891,630, 6,017,696 and International Application, Pub. No. WO 97/01646.
Electrical or electrochemical detection eliminates many of the disadvantages inherent in use of radioactive or fluorescent labels to detect interactions between the probe and target molecules. This process offers, for example, a detection technique that is safe, inexpensive, and sensitive, and is not burdened with complex and onerous regulatory requirements.
The development of microfabricated arrays of biomolecules (microarrays) has led to further improvements on traditional analytical techniques for the detection of molecular interactions between biomolecules. Microarrays of biomolecules (e.g., oligonucleotides, nucleic acids, proteins, peptides, or antibodies) have utility in a wide variety of applications in which molecular interactions between target molecules in a reaction mixture and large numbers of distinct probe molecules bound to defined regions of a substrate can be simultaneously assayed using electrical, optical, or radioactive detection strategies. Microarrays, therefore, satisfy the demand for inexpensive, high-throughput detection of biomolecular interactions.
Although microarrays have proven to be useful for high-throughput detection of molecular interactions between biomolecules, microarrays have proven to be inefficient with respect to reaction time. The probability that a particular target molecule will bind to an immobilized probe molecule is determined by the concentration of the target molecule in a reaction mixture, the diffusion rate of the target molecule, and the binding affinity of the target molecule for the immobilized probe molecule. Since target molecules in many diagnostic assays are only present in minute quantities (e.g.,  less than 10xe2x88x9212 M), interactions between target molecules and immobilized probe molecules therefore occur infrequently, and reaction times of several days are not uncommon.
In recognition of the inherent inefficiency of microarrays for detecting interactions between biomolecules, several techniques for increasing the rate of molecular interactions between biomolecules (termed xe2x80x9cbio-conjugation eventsxe2x80x9d herein) have been proposed. These techniques typically involve attaching a probe molecule to an electrode, which is then electrically biased to attract the target molecule through enhancing ion migration transport. For example, U.S. Pat. No. 5,653,939 to Hollis et al. discloses a method for detecting hybridization between a target molecule in a sample solution and an oligonucleotide probe bound at a test site, wherein an electric potential is applied to an electrode comprising the test site. However, current flow through the electrode causes electrolysis at the test site, which will reduce rather than enhance bio-conjugation events at such sites.
U.S. Pat. Nos. 5,605,662; 5,632,957; and 6,017,696 to Heller et al. disclose methods for controlling molecular biological reactions in microscopic formats that utilize a self-addressable, self-assembling microelectronic apparatus. Heller et al. further provide an apparatus in which target molecules labeled with fluorescent dyes are transported by free field electrophoresis to specific test sites where the target molecules are concentrated thereby, and reacted with specific probes bound to that test site. Unbound or non-specifically interacting target molecules are thereafter removed by reversing the electric polarity at the test site. Interactions between probe and target molecules are subsequently assayed using optical means. Heller et al., however, do not suggest using their devices for electrical or electrochemical detection of molecular interactions.
There remains a need in the art to develop more efficient, high-throughput devices and methods for the detection of molecular interactions between biomolecules. In particular, there remains a need in the art to develop more efficient, high-throughput devices and methods for electrical or electrochemical detection of molecular interactions. More particularly, there remains a need in the art to develop electrical or electrochemical detection devices in which the occurrence of a desired bio-conjugation event at a test site can be favorably manipulated, thereby yielding a device with increased throughput. The development of such devices, and methods for their use, would have wide application in the medical, genetic, and molecular biological arts.
The present invention provides an apparatus and methods for efficient, high-throughput detection of bio-conjugation events occurring at a test site. The invention specifically provides apparatuses comprising a pair of manipulation electrodes at a test site to enhance occurrence of said bio-conjugation events. In particular embodiments, the apparatuses of the invention also comprise detection electrodes at a test site for electrical or electrochemical detection of bio-conjugation events occurring at the test site. In the apparatuses of the invention, the detection and manipulation electrodes are separate and distinct, and are arranged in the apparatus to avoid electrolysis at the test site. In one embodiment of the apparatuses of the invention, the arrangement of the manipulation electrodes is such that electrolysis is prevented from occurring at the manipulation electrodes. In another particular embodiment of the invention, the arrangement of the manipulation electrodes in the apparatus is such that electrolysis is allowed to occur at the manipulation electrodes.
One apparatus of the present invention comprises a supporting substrate, a test site electrode in contact with the supporting substrate, a plurality of linker moieties in contact with the test site electrode and to which probe molecules are immobilized, a pair of manipulation electrodes oriented with respect to the test site electrode to be capable of generating an electrical potential at the test site electrode and where at least one manipulation electrode is in contact with the sample solution, at least one counter-electrode in electrochemical contact with the test site electrode, a means for producing an electrical signal at the test site electrode, a means for detecting changes in the electrical signal at each test site electrode, a means for producing an electrical potential between the manipulation electrodes, and an electrolyte solution in contact with the test site electrode, linker moiety, probes, counter-electrode, and at least one of the manipulation electrodes. The test site electrode and counter-electrode are operated in a manner that prevents current flow through said electrodes, thereby avoiding electrolysis therein upon application of an electrical potential between the electrodes.
Another apparatus of the present invention comprises a supporting substrate, a plurality of test site electrodes in contact with the supporting substrate, a plurality of linker moieties in contact with the test site electrodes and to which probe molecules are immobilized, a plurality of manipulation electrode pairs oriented with respect to the test site electrodes to be capable of generating an electrical potential at the test site electrodes and where at least one manipulation electrode in each pair is in contact with the sample solution, at least one counter-electrode in electrochemical contact with the test site electrodes, a means for producing an electrical signal at each test site electrode, a means for detecting changes in the electrical signal at each test site electrode, a means for producing an electrical potential between each manipulation electrode pair, and an electrolyte solution in contact with the test site electrodes, linker moieties, probes, counter-electrode, and at least one of the manipulation electrodes in each manipulation electrode pair. The test site electrodes and counter-electrode are operated in a manner that prevents current flow through said electrodes, thereby avoiding electrolysis therein upon application of an electrical potential between the electrodes. Preferably, the probe molecules immobilized at any particular test site are identical to each other, while each test site comprises probe molecules unique to that test site.
Another apparatus of the present invention comprises a supporting substrate, a test site electrode in contact with the supporting substrate, a plurality of linker moieties in contact with the test site electrode and to which probe molecules are immobilized, a pair of manipulation electrodes oriented with respect to the test site electrode to be capable of generating an electrical potential that does not terminate at the test site electrode and where at least one manipulation electrode is in contact with the sample solution, at least one counter-electrode in electrochemical contact with the test site electrode, a means for producing an electrical signal at the test site electrode, a means for detecting changes in the electrical signal at each test site electrode, a means for producing an electrical potential between the manipulation electrodes, and an electrolyte solution in contact with the test site electrode, linker moiety, probes, counter-electrode, and at least one of the manipulation electrodes. The test site electrode and counter-electrode are operated in a manner that prevents current flow through said electrodes, thereby avoiding electrolysis therein upon application of an electrical potential between the electrodes.
Another apparatus of the present invention comprises a supporting substrate, a plurality of test site electrodes in contact with the supporting substrate, a plurality of linker moieties in contact with the test site electrodes and to which probe molecules are immobilized, a plurality of manipulation electrode pairs oriented with respect to the test site electrodes to be capable of generating an electrical potential that does not terminate at the test site electrodes and where at least one manipulation electrode in each pair is in contact with the sample solution, at least one counter-electrode in electrochemical contact with the test site electrodes, a means for producing an electrical signal at each test site electrode, a means for detecting changes in the electrical signal at each test site electrode, a means for producing an electrical potential between each manipulation electrode pair, and an electrolyte solution in contact with the test site electrodes, linker moieties, probes, counter-electrode, and at least one of the manipulation electrodes in each manipulation electrode pair. The test site electrodes and counter-electrode are operated in a manner that prevents current flow through said electrodes, thereby avoiding electrolysis therein upon application of an electrical potential between the electrodes. Preferably, the probe molecules immobilized at any particular test site are identical to each other, while each test site comprises probe molecules unique to that test site.
In some apparatuses of the present invention, the manipulation electrodes are arranged so as to prevent electrolysis from occurring at the manipulation electrodes. In other specific apparatuses of the invention, the arrangement of the manipulation electrodes is such that electrolysis is allowed to occur at the manipulation electrodes.
The apparatus of the present invention may further comprise a plurality of wells wherein each well encompasses a test site electrode to which is attached a linker moiety which is further attached to a probe molecule, at least one of the manipulation electrodes in each manipulation electrode pair, and at least one counter-electrode that is sufficient to interrogate the test site electrode in contact with linker moiety and probe molecules. The apparatus of the present invention may also further comprise at least one reference electrode. Preferably, the probe molecules in any particular well are identical to each other, while each well comprises probe molecules unique to that well.
The present invention provides methods employing the apparatus which are useful for the electrical or electrochemical detection of molecular interactions between probe molecules immobilized on (or through) linker moieties in contact with test site electrodes and target molecules in a sample solution. In one method of the present invention, a first electrical signal is detected at a test site electrode in contact with linker moieties to which probe molecules have been immobilized. Thereafter, the test site electrode is exposed to a sample mixture containing a particular target molecule, and an electrical potential is applied between a pair of manipulation electrodes oriented with respect to the test site electrode to generate an electrical potential at the test site electrode that enhances concentration of the target molecules at the test site. Preferably, the polarity of the electrical potential applied to the pair of manipulation electrodes is periodically alternated during the period that a potential is applied between the manipulation electrodes. A second electrical signal is then detected at the test site electrode in the absence of a potential at the manipulation electrodes and after a time sufficient for a biomolecular interaction to have occurred between the probe molecules immobilized at the test site electrode and target molecules in the sample mixture. The first and second electrical signals are compared, and molecular interactions between immobilized probe molecules and target molecules in the sample mixture are detected by determining that the first electrical signal is different from the second electrical signal.
In another method of the present invention, a first electrical signal is detected at a test site electrode in contact with linker moieties to which probe molecules have been immobilized. The test site electrode is exposed to a sample mixture containing a particular target molecule, an electrical potential is applied between a pair of manipulation electrodes oriented with respect to the test site electrode to generate an electric potential that does not terminate at the test site electrode, and the polarity of the electrical potential applied to the manipulation electrode pair is periodically alternated. Thereafter, a second electrical signal is detected at the test site electrode in the absence of a potential at the manipulation electrodes and after a time sufficient for a biomolecular interaction to have occurred between the probe molecules immobilized at the test site electrodes and target molecules in the sample mixture. The first and second electrical signals are then compared, and molecular interactions between immobilized probe molecules and target molecules in the sample mixture are detected by determining that the first electrical signal is different from the second electrical signal.
In still other embodiments of the methods of the present invention, target molecules in a sample mixture are labeled with an electrochemically-active reporter molecule prior to exposing the sample mixture to the test site electrode.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.